The invention is related to a factor which potentiates the pesticidal activity of a Bacillus related pesticide, a chemical pesticide and/or a virus with pesticidal properties, as well as methods for obtaining the factor. Furthermore, the invention relates to pesticidal compositions comprising the factor and a pesticidal carrier, or the factor and a Bacillus related pesticide, a chemical pesticide and/or a virus with pesticidal properties as well as methods of using such compositions. The invention further relates to a mutant or variant Bacillus strain in which such a factor is obtained in larger amounts or has a greater potentiating activity compared to the parental strain and methods for producing such mutant or variant strains.
Every year, pests detrimental to agriculture, forestry, and public health cause losses in the millions of dollars. Various strategies have been used to control such pests.
One strategy is the use of chemical pesticides with a broad range or spectrum of activity. However, there are a number of disadvantages with using chemical pesticides. Specifically, because of their broad spectrum of activity, these pesticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests. Additionally, chemical pesticides are frequently toxic to animals and humans. Furthermore, targeted pests frequently develop resistance when repeatedly exposed to such substances.
Another strategy involves the use of biopesticides to control insect, fungal and weed infestations. Biopesticides are naturally occurring pathogens and/or the substances produced by these pathogens. The advantage of using biopesticides is that they are generally less harmful to non-target organisms and the environment as a whole compared to chemical pesticides.
The most widely used biopesticide is Bacillus thuringiensis. Bacillus thuringiensis is a motile, rod-shaped, gram-positive bacterium that is widely distributed in nature, especially in soil and insect-rich environments. During sporulation, Bacillus thuringiensis produces a parasporal crystal inclusion(s) which is insecticidal upon ingestion to susceptible insect larvae of the orders Lepidoptera, Diptera, and Coleoptera. The inclusions may vary in shape, number, and composition. They are comprised of one or more proteins called delta-endotoxins, which may range in size from 27-140 kDa. The insecticidal delta-endotoxins are generally converted by proteases in the larval gut into smaller (truncated) toxic polypeptides, causing midgut destruction, and ultimately, death of the insect (Hxc3x6fte and Whiteley, 1989, Microbiological Reviews 53:242-255).
There are several Bacillus thuringiensis strains that are widely used as biopesticides in the forestry, agricultural, and public health areas. Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. aizawai produce delta-endotoxins specific for Lepidoptera. A delta-endotoxin specific for Coleoptera is produced by Bacillus thuringiensis subsp. tenebrionis (Krieg et al., 1988, U.S. Pat. No. 4,766,203). Furthermore, Bacillus thuringiensis subsp. israelensis produces delta-endotoxins specific for Diptera (Goldberg, 1979, U.S. Pat. No. 4,166,112).
Other Bacillus thuringiensis strains specific for dipteran pests have also been described. A Bacillus thuringiensis isolate has been disclosed which is toxic to Diptera and Lepidoptera (Hodgman et al., 1993, FEMS Microbiology Letters 114:17-22). Sodium dodecyl polyacrylamide gel electrophoresis of the purified crystal delta-endotoxin from this isolate revealed three protein species which are related to CryIA(b), CryIB, and CryIIA toxins. There has also been disclosed a Bacillus thuringiensis isolate which produces a dipteran-active crystal comprised of proteins with molecular weights of 140, 122, 76, 72, and 38 kDa (Payne, 1994, U.S. Pat. No. 5,275,815). EPO 480,762 discloses five B.t. strains which are each active against dipteran pests; each also have a unique crystal delta-endotoxin pattern.
Several Bacillus thuringiensis strains have been described which have pesticidal activity against pests other then Lepidoptera, Coleoptera, and Diptera. Five Bacillus thuringiensis strains have been disclosed which produce delta-endotoxins that are toxic against nematodes (Edwards, Payne, and Soares, 1988, Eur. Pat. Appl. No. 0 303 426 B1). There has also been disclosed a Bacillus thuringiensis strain, PS81F, which can be used to treat humans and animals hosting parasitic protozoans (Thompson and Gaertner, 1991, Eur. Pat. Appl. No. 0 461 799 A2). Several Bacillus thuringiensis isolates have also been disclosed with activity against acaride pests. These isolates produce crystals comprised of proteins with molecular weights in the (wide) range of 35 kDa to 155 kDa (Payne, Cannon, and Bagley, 1992, PCT Application No. WO 92/19106). There have also been disclosed Bacillus thuringiensis strains with activity against pests of the order Hymenoptera (Payne, Kennedy, Randall, Meier, and Uick, 1992, Eur. Pat. Appl. No. 0 516 306 A2); with activity against pests of the order Hemiptera (Payne and Cannon, 1993, U.S. Pat. No. 5,262,159); with activity against fluke pests (Hickle, Sick, Schwab, Narva, and Payne, 1993, U.S. Pat. No. 5,262,399; and with activity against pests of the order Phthiraptera (Payne and Hickle, 1993, U.S. Pat. No. 5,273,746). Furthermore, another strain of Bacillus thuringiensis subsp. kurstaki, WB3S-16, isolated from Australian sheep wool clippings, has been disclosed that is toxic to the biting louse Damalinia ovis, a Phthiraptera pest (Drummond, Miller, and Pinnock, 1992, J. Invert. Path. 60:102-103).
The delta-endotoxins are encoded by cry (crystal protein) genes which are generally located on plasmids. The cry genes have been divided into six classes and several subclasses based on relative amino acid homology and pesticidal specificity. The major classes are Lepidoptera-specific (cryI); Lepidoptera-and Diptera-specific (cryII); coleoptera-specific (cryII); Diptera-specific (cryIV) (Hxc3x6fte and Whiteley, 1989, Microbiological Reviews 53:242-255); Coleoptera- and Lepidoptera-specific (referred to as cryV genes by Tailor et al., 1992, Molecular Microbiology 6:1211-1217); and Nematode-specific (referred to as cryV and cryVI genes by Feitelson et al., 1992, Bio/Technology 10:271-275).
Delta-endotoxins have been produced by recombinant DNA methods. The delta-endotoxins produced by recombinant DNA methods may or may not be in crystal form.
Some strains of Bacillus thuringiensis have been shown to produce a heat-stable pesticidal adenine-nucleotide analog, known as xcex2-exotoxin type I or thuringiensin, which is pesticidal alone (Sebesta et al., in H. D. Burges (ed.), Microbial Control of Pests and Plant Diseases, Academic Press, New York, 1980, pp. 249-281). xcex2-exotoxin type I has been found in the supernatant of some Bacillus thuringiensis cultures. It has a molecular weight of 701 and is comprised of adenosine, glucose, and allaric acid (Farkas et al., 1977, Coll. Czechosslovak Chem. Comm. 42:909-929; Lxc3xcthy et al., in Kurstak (ed.), Microbial and Viral Pesticides, Marcel Dekker, New York, 1982, pp. 35-72). Its host range includes, but is not limited to, Musca domestica, Mamestra configurata Walker, Tetranychus urticae, Drosophila melanogaster, and Tetranychus cinnabarinus. The toxicity of xcex2-exotoxin type I is thought to be due to inhibition of DNA-directed RNA polymerase by competition with ATP. It has been shown that xcex2-exotoxin type I is encoded by a cry plasmid in five Bacillus thuringiensis strains (Levinson et al., 1990, J. Bacteriol. 172:3172-3179). xcex2-exotoxin type I was found to be produced by Bacillus thuringiensis subsp. thuringiensis serotype 1, Bacillus thuringiensis subsp. tolworthi serotype 9, and Bacillus thuringiensis subsp. darmstadiensis serotype 10.
Another xcex2-exotoyin classified as xcex2-exotoxin type II has been described (Levinson et al., 1990, J. Bacteriol. 172:3172-3179). xcex2-exotoxin type II was found to be produced by Bacillus thuringiensis subsp. morrisoni serotype 8ab and is active against Leptinotarsa decemlineata. The structure of xcex2-exotoxin type II is not completely known, but is significantly different from that of xcex2-exotoxin type I in that a pseudouridine moiety is in the place of adenine in which attachment to the ribose ring is at a position that would otherwise be occupied by a proton (Levinson, in Hickle and Finch (eds.), Analytical Chemistry of Bacillus thuringiensis, ACS Symposium Series, Washington, D.C., 1990, pp. 114-136). Furthermore, there is only one signal in the proton NMR spectrum corresponding to the nucleoside base (at 7.95 ppm), and does not have a ribose-type anomeric protein signal (5.78 ppm).
Other water soluble substances that have been isolated from Bacillus thuringiensis include alpha-exotoxin which is toxic against the larvae of Musca domestica (Luthy, 1980, FEMS Microbiol. Lett. 8:1-7); gamma-exotoxins, which are various enzymes including lecithinases, chitinases, and proteases, the toxic effects of which are expressed only in combination with beta-exotoxin or delta-endotoxin (Forsberg et al., 1976, Bacillus thuringiensis: Its Effects on Environmental Quality, National Research Council of Canada, NRC Associate Committee on Scientific Criteria for Environmental Quality, Subcomittees on Pesticides and Related Compounds and Biological Phenomena); sigma exotoxin which has a structure similar to beta-exotoxin, and is also active against Leptinotarsa decemlineata (Argauer et al., 1991, J. Entomol. Sci. 26:206-213); and anhydrothuringiensin (Prystas et al., 1975, Coll. Czechosslovak Chem. Comm. 40:1775).
The art has strived to achieve increased mortality of B.t. formulations. Means have included searching for new strains with increased mortality, attempting to engineer present strains, and attempting to design more effective formulations by combining B.t. spores and crystals with new pesticidal carriers chemical pesticides, or enhancers (see, for example, U.S. Pat. No. 5,250,515, a trypsin inhibitor). It is therefore an object of the present invention to potentiate the pesticidal activity of pesticides.
The invention is related to a novel factor which unlike factors known in the art potentiates the pesticidal activity of a Bacillus related pesticide. Specifically, the factor of the present invention is a potentiator. As defined herein, a xe2x80x9cpotentiatorxe2x80x9d is a substance which has no significant pesticidal activity, e.g. having an LC50 (LC50 is the concentration of the substance required to kill 50% of the pests) of more than about 3000 xcexcg/g as assayed by bioassay (see Section 6) but acts to increase the pesticidal activity of a Bacillus related pesticide at least about 50% and does not cause larval stunting. As noted in Section 2, other substances capable of enhancing pesticidal activity known in the art such as delta-endotoxins, trypsin inhibitors and exotoxins have pesticidal activity.
In a specific embodiment, the factor is water soluble. As defined herein, a substance or compound is xe2x80x9cwater solublexe2x80x9d if at least about 1 mg of a substance can be dissolved in 1 ml of water. The factor may also potentiate the pesticidal activity of a chemical pesticide and/or a virus with pesticidal properties.
As defined herein, xe2x80x9ca Bacillus related pesticidexe2x80x9d is a Bacillus (e.g. Bacillus thuringiensiss or Bacillus subtilis) starin, spore, or substance, e.g. protein or fragment thereof having activity against or which kill pests or a microorganism capable of expressing a Bacillus gene encoding a Bacillus protein or fragment thereof having activity against or which kill pests (e.g. Bacillus thuringiensis delta-endotoxin) and an acceptable carrier (see Section 5.2., infra, for examples of such carriers). The pest may be, for example, an insect, a nematode, a mite, or a snail. A microorganism capable of expressing a Bacillus gene encoding a Bacillus protein or fragment thereof having activity against or which kill pests inhabits the phylloplane (the surface of the plant leaves), and/or the rhizosphere (the soil surrounding plant roots), and/or aquatic environments, and is capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms and provide for the stable maintenance and expression of a Bacillus gene encoding a Bacillus protein or fragment thereof having activity against or which kill pests. Examples of such microorganisms include but are not limited to bacteria, e.g. genera Bacillus, Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, Alcaligenes, and Clostridium; algae, e.g. families Cyanophyceae, Prochlorophyceae, Rhodophyceae, Dinophyceae, Chrysophyceae, Prymnesiophyceae, Xanthophyceae, Raphidophyceae, Bacillariophyceae, Eustigmatophyceae, Cryptophyceae, Euglenophyceae, Prasinophyceae, and Chlorophyceae; and fungi, particularly yeast, e.g. genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.
As defined herein, xe2x80x9cpesticidal activityxe2x80x9d measures the amount of activity against a pest through killing or stunting of the growth of the pest or protecting the plant from pest infestation.
The invention further relates to pesticidal compositions comprising the factor and a pesticidal carrier as well as the factor and a Bacillus related pesticide, chemical pesticide and/or a virus with pesticidal properties.
The invention further relates to methods of using the pesticidal compositions of the present invention. In one embodiment, the invention relates to a method for controlling a pest comprising exposing the pest to a pest-controlling amount of the composition. In another embodiment, the invention relates to a method for decreasing the resistance of a pest to a Bacillus related pesticide comprising exposing the pest to a composition comprising the factor and a pesticidally acceptable carrier. The invention further relates to a method for potentiating the pesticidal activity of a Bacillus related pesticide comprising administering to a pest exposed to a Bacillus related pesticide a composition comprising the factor and carrier in amount effective to potentiate the pesticidal activity of a Bacillus related pesticide.